1. Field of the Invention
The present invention relates generally to apparatus for performing electrophoresis, and in particular to improved materials and methods for coating electrophoresis tubes to inhibit zone distortion resulting from electroendoosmosis and adsorption of solutes on the inner surface of the tube.
2. Description of the Background Art
Electrophoretic separation techniques, including zone electrophoresis, displacement electrophoresis (isotachophoresis), and isoelectric focusing may be performed in a variety of apparatus. Of interest to the present invention is the use of thin-walled, narrow-bore capillary tubes for containing the carrier-free separation medium. Such tubes are particularly useful since zone deformation caused by the evolution of heat during the electrophoretic separation is negligible.
The use of capillary tubes in electrophoresis techniques, however, results in zone deformation arising from other mechanisms, including both electroendosmosis and adsorption of the substances to be separated resulting, for example, from interaction of the substance with charges on the inner tube surface. Zone deformation is a particular problem with macromolecular substances, such as proteins, which possess multiple binding sites capable of interacting strongly with the inner wall of the tube. The problem is acute in zone electrophoresis where the zones are not as well defined as with other techniques, such as isoelectric focusing, where the zones are substantially sharpened.
The problems associated with adsorption and electroendoosmosis have been largely overcome by coating the inner wall of the electrophoresis tube with various polymeric substances. One of the most successful techniques has been the use of methylcellulose as described by Hjerten (1967) Chromatogr. Rev. 9:122-219. The methylcellulose is applied by baking at an elevated temperature and low pH in the presence of formaldehyde. Although successful, the method suffers from a number of drawbacks. The methylcellulose is adsorbed to the glass (quartz) surface and must therefore be renewed after about five days. Moreover, the thickness of the coating is non-uniform so that a relatively thick application is required to assure that the entire area is sufficiently covered. Also, it is difficult to work with the methylcellulose since it is highly viscous. For instance, due to the high viscosity, it is virtually impossible to fill a capillary electrophoresis tube with an inner diameter as small as 0.05 mm with the methylcellulose solution, which is one step in the coating procedure. Finally, in both the methylcellulose coated tubes and in the tubes coated as described herein the viscosity is higher at the wall than at the axis of the tube--a condition that eliminates electroendosmosis as shown by Hjerten, supra. This difference in viscosity causes the solutes to migrate more slowly at the wall than at the axis of the tube which is equivalent to a zone deformation. However, since the tubes coated as described herein have a very thin monomolecular coating the zone deformation is negligible, while the methylcellulose-coated tubes have a much thicker coating and therefore may cause an observable zone deformation, particularly when the tubes are very narrow since the coating then will represent percentually a large part of the tube volume.
Other coating materials have also been proposed for electrophoretic applications. Jorgensen et al. (1983) Science 222:266-272, describe the use of silica tubes modified with glycol groups to decrease the adsorption of proteins. The reported results clearly indicate that electroendoosmosis in such tubes is still very pronounced and therefore also the adsorption. Radola (1980) Electrophoresis 1:43-56, describes isoelectric focusing using gels of cross-linked polyacrylamide covalently bound to glass plates or polyester films pretreated with .gamma.-methacryloxypropyltrimethoxysilane. The purpose of this pretreatment is only to fix the gel to the glass or the polyester. In that technique the gel fills the whole separation chamber. The separations are accordingly not performed in free solution as is the case with the present invention and therefore no problems with electroendosmosis occur.